Plate and method for high throughput screening

ABSTRACT

A multiple well plate and method for media exchange, including a body defining a plurality of cell wells each connected via a channel to one of a plurality of aspiration holes, is provided. The cell wells contain a porous, hydrophilic frit which is suspended on a ledge above a reservoir of fluid media and supports a tissue sample. The properties of the frit wick the fluid media upwards to supply the tissue sample with nutrients for growth and proliferation. Old media is aspirated from the wells by a liquid handling device which inserts a pipette tip into the aspiration holes. The pipette tip applies a suction pressure which draws the media out of the cell well, through the channel, into the aspiration hole and out through the pipette tip. New media is dispersed through the pipette tip and directly into the cell well.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.10/157,562, filed May 29, 2002, which claims the benefit of U.S.Provisional Application No. 60/294,430, filed May 30, 2001, both ofwhich are hereby incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to devices and methods for promoting thegrowth of tissue in experimental and production settings, moreparticularly the use of specialized plates to house the tissue and thecycling of media to biologically sustain the tissue.

BACKGROUND OF THE INVENTION

High-throughput screening typically requires parallel processing ofbatches of samples, typically in multiple well plates (MWPs) of 24, 48,96, and 384, or more, wells per plate. MWPs are standard sizes that canbe used with existing high-throughput machinery, such as withrobotic-controlled pipetters. Each pipetting station of a roboticcontrolled pipetter employs pipetting heads having an array of pipettetips that address multiple wells simultaneously. Although usedeffectively for the screening of liquid samples, the current multiplewell plates are generally ineffective for screening plant and othertissues, and the secretory products associated with these tissues, thatrequire, or prefer, more complex environments such as solid supportstructures.

For example, attempts have been made to grow plants in MWPs bysuspending the plants in a liquid media within each well. However, theplant tissue is deprived of oxygen when sitting in the liquid,effectively “drowning” the plant tissue in an anaerobic environment.Other attempts have been made using media that are generally more solidand provide a substrate on which the plant tissue may be supported abovethe fluid, such as a gel or filter paper disk. Although these types ofsupports avoid drowning the plants, they are difficult to exchange andreplenish when the nutrients or media have been depleted. Paper bridgesdoused in liquid media have also been used as tissue supports and theliquid media is somewhat more easily replenished. However, empiricalevidence has shown that paper bridges are difficult to manage in anautomated system and are generally ineffective at promoting plant tissuegrowth. Without being wed to any particular theory, this may be becausethe liquid media does not easily penetrate the paper bridge (i.e., thepaper bridge is only mildly hydrophilic) and the tissue supportedthereon lacks a continuous supply of media.

A common approach to supplying fresh media to plant tissue is to movethe plant tissue to a container holding fresh media. Movement of theplant tissue is a relatively slow and labor intensive process, asmultiple plates must be replenished and otherwise prepared for eachbatch of plant tissue. In addition there is a threat of loss orcontamination of the tissue samples when they are removed from thewells.

Another approach to aspirating and removing spent media and tissuebyproducts is to use an assay plate having a plurality of wells, witheach well having a hole or port at the base of the well. A filter ispositioned at the bottom of each well to support the tissue. Spent mediacan be vacuum harvested from each well through the port using a vacuummanifold assembly. One example of a vacuum manifold assembly is theMultiScreen Vacuum Manifold system manufactured by MILLIPORE of Bedford,Mass. The assay plate rests on a manifold that supplies a vacuum whichdraws the media through the filter disks, out of the cell wells andthrough the ports, where it is captured in the manifold below. Althoughthe filter disks in the assay plate allow media to be drawn out of theplate, it is difficult for the filter disks to retain enough media tosupport tissue maintenance and growth for any length of time. Becausethe ports at the bottom of the wells are open to the ambient air, theports may allow media to leak or evaporate and may also provide a pathfor microbial contamination of the wells. In addition, the wells of theassay plate cannot be individually sampled because the vacuum manifoldharvests the media from all of the wells at once.

The tissue of animals, and other types of organisms may also require, orprefer, solid support structures that inhibit the use of multiple wellplates and high-throughput screening techniques. For instance, thegrowth of cartilage cells may be promoted by the use of a collagenfibril matrix that simulates an in vivo environment. Similar to theplant tissue discussed above, the cartilage cells need a supply of freshmedia that is replenished at various intervals to survive and/orproliferate. In addition, some of the cartilage cells proliferate withinthe collagen fibril matrix and cannot be moved independent of thematrix. Moving the cells to a new plate with a fresh supply of mediarequires movement of the entire collagen matrix which is a relativelyslow and inefficient process that exposes the tissue to contamination.

It would be advantageous to have a multiple well plate that allows theuse of high throughput screening methods for non-liquid samples. Inaddition, it would be advantageous to have a multiple well plate thatallows the use of high throughput screening methods for tissues thatrequire, or prefer, solid support structures. It would be furtheradvantageous to have a multiple well plate that promotes the growth oftissue, such as plant tissue, without posing the risk of drowning thetissue in liquid media or allowing the tissue to dehydrate or becomecontaminated. It would also be advantageous to have a multiple wellplate that allows media to be easily replenished, without unduedisturbance of the tissue contained in the wells. Additionally, it wouldbe advantageous to have the capability of sampling less than the totalnumber of wells in the plate without disturbing the unsampled wells.

SUMMARY OF THE INVENTION

The present invention addresses the above needs and achieves otheradvantages by providing a multiple well plate (MWP) and method for mediaexchange that promotes the growth of plant tissue, and other types oftissue, by controlling the supply of media to the tissue and allowingfor the regular exchange (removal and addition) of media withoutdisturbing the tissue. The MWP includes an array of wells, with eachwell being coupled with an adjacent aspiration hole that allows media tobe aspirated from the wells using a conventional, automated pipettehead. The MWP and pipette head provide a virtually complete exchange ofthe spent media because of the novel dual-well architecture. Ahydrophilic, porous frit housed within each well supports the tissue andholds the media in its interstices, allowing contact between the tissueand the media while avoiding an anaerobic condition. The media is wickedupwards in sufficient quantities to provide nutrients to the tissue andpromote proliferation of the tissue.

In one embodiment, the invention includes a plate for holding a porousfrit that supports a tissue. The porous frit is saturated in a mediathat may be regularly aspirated and refreshed, for example, by atop-loading pipette device. The plate comprises a body with an uppersurface defining a first hole and a second hole. The first hole has afirst hole upper edge defined by the upper surface of the body and afirst hole bottom portion defined within the body and below the uppersurface of the body. The first hole is configured to receive the porousfrit and the tissue, and to hold the media bathing the porous frit andthe tissue. The second hole has a second hole upper edge defined by theupper surface of the body and a second hole bottom portion definedwithin the body and below the upper surface of the body. The second holebottom portion is in fluid communication with the first hole bottomportion so that the pipette device can access the second hole upper edgeto aspirate the media by applying a vacuum. The pipette device alsorefreshes the media by adding fresh media directly onto the frit in eachwell.

In another aspect, the body of the plate further defines a passageconnecting the first hole bottom portion and the second hole bottomportion in fluid communication. The body may further include a ledgeprotruding into the first hole bottom portion for supporting the fritabove a reservoir of fluid. The body may also define a plurality of thefirst and second holes, with each first hole in fluid communication witha respective one of the second holes to form a MWP. In another aspect,the body defines an array of first and second holes, for example 12, 24,48, 96, 384, or 1536 first and second holes, wherein each first hole isin fluid communication with a respective one of the second holes. Thefirst and second holes preferably have cylindrical shapes.

In yet another aspect, the top surface of the body is configured toreceive a cover plate disposed thereon. Preferably the cover plate istransparent to light transmission and the first hole upper edge isconfigured to also allow light transmission, thereby promoting planttissue growth.

In another embodiment, the present invention includes a method of makinga plate for holding a porous frit that supports a tissue wherein theporous frit is saturated in a media. The media is regularly aspiratedand refreshed by a pipette device to promote proliferation of thetissue. The method includes providing a body with an upper surface anddefining a first and second holes in the body. Defining a first hole inthe body includes drilling through the upper surface of the body to forma first hole upper edge and drilling below the upper surface to form afirst hole bottom portion. The second hole is defined by drillingthrough the upper surface of the body to form a second hole upper edgeand drilling below the upper surface to form a second hole bottomportion. The first and second hole bottom portions are connected influid communication by forming a passage in the body and between thefirst hole bottom portion and the second hole bottom portion.Preferably, the passage is formed by inserting a saw disc into the firsthole bottom portion and moving the saw laterally until encountering thesecond hole bottom portion.

In yet another embodiment, the present invention includes a method ofusing a MWP. A frit is placed into each of the plurality of first holesand tissue, preferably a duckweed or other plant tissue, is placed ontothe frit. A media is dispensed into each of the plurality of firstholes. A pipette is inserted into each of the plurality of second holesand used to aspirate the media from the first holes. The media isaspirated by applying a suction pressure to the second holes using thepipette. The suction pressure draws the media from the first hole, intothe second hole and into the pipette so as to flush the media from theplate. Fresh media can be re-dispensed into the plurality of first holesafter aspirating the media from the second holes.

In still another embodiment, the present invention includes a fritmaterial for supporting a tissue having a porous structure, a topsurface and a bottom surface. The porous structure has hydrophilicproperties and a plurality of interstices. The top surface is configuredto support the tissue. The bottom surface is in fluid communication witha reservoir of fluid media. The hydrophilic properties of the porousstructure wick the fluid into its interstices so that the supportedtissue is supplied with sufficient liquid media from the reservoir topromote growth of the tissue.

The present invention has several advantages. For example, the tissuesamples in the wells do not have to be moved or disturbed when theprovided media is spent, cutting down on workload and ensuring sterileand optimal growth conditions. The plates may be used with conventionalliquid handling pipette heads of the fixed tip or individuallycontrollable tip versions because the aspiration holes are accessiblefrom the upper surface of the body, i.e., a top-loading arrangement. Theuse of robotic liquid handlers with the plate promotes a well-to-wellconsistency in the treatment of the tissue, as well as the efficientremoval and replacement of the media. The top-loading aspect allows theuse of a standard lid for sterility control and removes the need for aseparate vacuum manifold station for pulling out media. The lack of amanifold allows for the differential treatment of each well and providesflexibility in liquid handler design and selection, as well asexperimental model and sample interrogation functions. The liquid headcan be configured to remove the media as well as add new media with nochange of tooling or pipette tips.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will nowbe made to the accompanying drawings, which are not necessarily drawn toscale, and wherein:

FIG. 1 is a plan view of a MWP for liquid media exchange of a firstembodiment of the present invention;

FIG. 2 is an enlarged, cross-sectional view of a single well and anaspiration hole from the MWP of FIG. 1;

FIG. 3 is a perspective view of a tip seal of another embodiment of thepresent invention;

FIG. 4 is a perspective view of a system for replenishing media in theMWP shown in FIG. 1 of another embodiment of the present invention;

FIG. 5 is a flow chart of a method of replenishing media using thesystem of FIG. 4; and

FIG. 6 is a sectional view of the well and aspiration hole of FIG. 2being machined in another embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Likenumbers refer to like elements throughout.

A multiple well plate (MWP) 10 of the present invention includes a body12 having an upper surface 14. The body 12 defines an array of firstholes, or wells 16 and an array of second aspiration holes 22, as shownin FIG. 1. A plurality of channels 28 each connect a respective one ofthe wells 16 to an adjacent one of the aspiration holes 22, as shown inFIG. 2. In one embodiment, disposed in each of the wells 16 is a porous,hydrophilic frit 30 which supports a tissue sample 32 over a reservoirof liquid media 36. The porous, hydrophilic properties of the frit 30wick the media 36 upwards, so as to supply the media to the tissuesample 32. Exchange of old, depleted media 36 is facilitated by theaspiration holes 22 which are each sized and configured to receive apipette tip 34. During aspiration, several of the pipette tips areinserted into the aspiration holes 22 and apply a vacuum pressure. Thevacuum pressure draws the fluid media 36 out of each of the wells 16,through the channels 28, through the adjacent one of the aspirationholes 22 and into the pipette tips.

The tissue sample 32 is preferably a plant tissue, such as dicot andmonocot tissue such as tissue from corn (Zea mays), Brassica sp. (e.g.,B. napus, B. rapa, B. juncea), particularly those Brassica speciesuseful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryzasativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, and conifers. In some embodiments the tissue iscallus tissue from duckweed or gymnosperms. The present invention may beparticularly effective with plant tissues that thrive with minimalfluid, such as tissue derived from gymnosperms.

The body 12 of the MWP 10 is preferably constructed of a polycarbonateblock that can be machined and is resistant enough to heat to besterilized in an autoclave for reuse. Generally, the hardness ofpolycarbonate allows it to be machined by computer controlled millingmachine (CNC), or other automatic machining process, into complex,precision shapes. The body could also be constructed of other materials,such as a polystyrene, polysulphone, other synthetic materials, metals,ceramics, glass, etc. The body 12 is rectangular in shape, being5.03±0.01 inches in length, 3.365±0.01 inches in width and 0.813±0.01inches in height, a configuration compatible with most conventionalliquid handling machines. The body 12 includes other features such as abase 13 and a pair of 45° chamfers 15 on opposing corners of one widthof the body. The base 13 provides a ledge and can serve as a gripping ormounting surface in conventional equipment. The pair chamfers 15 canserve as reference marks to ensure the proper orientation of the body,especially when it is important to keep track of the location of eachwell. The body 12 also has a flat, upper surface 14 through which theholes 16 and 22 are drilled. It should be noted, that although the sizeof the body is preferably configured for compatibility with preexistingequipment, the dimensions of the body can be varied as desired.

The number, dimensions and locations of the wells 16 are also tailoredto be compatible with preexisting equipment. For instance, the platepreferably has 24 wells in an array of 4 by 6, or 48 wells in an arrayof 6 by 8 to be compatible with most liquid handling devices. Other welldensities could be used such as 6 wells, or 96 wells that are compatiblewith conventional devices. However, nonstandard well densities couldalso be used, such as single well or a 1000 wells. Generally the numberof wells will be limited by such practicalities as the size of the body12, the type of tissue being grown, the capabilities of the equipmentusing the wells and the size of the wells themselves.

Preferably, each of the wells 16 has a standard cylindrical shape with adiameter of 0.62 inches and a depth of 0.60±0.01 inches for the 24 wellplate. The diameter of each well can be varied as desired, and is basedon several factors, such as the initial size of the tissue to be placedin the well, the growth rate of the tissue, and the length of time thetissue is to be propogated in the well before removal. Acenter-to-center distance between the wells is 0.76 inches for the 24well plate to ensure that the arrangement and motion of standard,automated pipette devices is compatible. The 24 well plate has beendetermined by the inventors to be particularly suitable to the tissuepropagation of duckweed callus, presenting a preferred balance of tissue32 volume and density of wells. The density of the cell wells 16 usedfor duckweed is preferably 96 wells or less due to the size of callusand rapid cell growth. Of course, other arrangements could also be usedfor more customized equipment, if desired.

The aspiration holes 22 extend through the upper surface 14 of the body12. Each of the 24 aspiration holes 22 is preferably adjacent to, andconnected in fluid communication with, a respective one of the wells 16.The pairing arrangement of the aspiration holes 22 and the wells 16allows aspiration without cross-contamination of the samples, such asoccurs with the prior art manifold well plates. In addition, the pairingarrangement allows the media of individual wells 16 of interest to beaspirated and refreshed selectively. Individual wells 16 could beaddressed selectively by hand or by automated equipment that allows theoperation of a single pipette independently of the other pipettes in thehead. Selectively addressing wells would be useful if, for instance, thetissue in one of the wells was generating a strong expression responseto an agent, such as increased growth of tissue, increased expression ofa polypeptide, added resistance to a selective agent such as aherbicide, or resistance to a plant pathogen. Other biochemical orbiophysical responses could also be assayed in individual wells with thepresent invention. The media from this well could be aspirated andtested more frequently than the other wells. Among other advantages,more frequent collection and testing of media from that well wouldprovide a stronger statistical correlation. If contamination is less ofa concern, each of the aspiration holes 22 could be paired with severalwells 16 to cut down on the number of aspiration iterations.

Each of the aspiration holes 22 are also preferably cylindrical in shapewith a depth of 0.66±0.01 inches and a diameter of 0.167 inches, so asto be able to receive a standard sized pipette tip 34 through its upperedge 24. Larger or smaller diameters, and different center-to-centerdistances could be used, depending upon the size of the pipette tip tobe inserted therein. Of course, the other shapes and other dimensionscould be varied to suit a customized arrangement, or other standardpipette shapes and lengths that are known to those of skill in the art.Cylindrical aspiration holes 22 are also preferable in that they areeasier to machine with rotating drill bits. The center-to-centerdistance between adjacent ones of the aspiration holes 22 is preferablythe same as the center-to-center distance between the wells 16, which is0.76 inches in the illustrated embodiment of 24 wells. The placement ofthe aspiration holes through the upper surface 14 of the body 12 and thesame center-to-center distance ensures that the same pipette headconfiguration can be used for aspiration of the media 36 as for thedispersion of the media.

Each of the channels 28 connects a respective pair of the cell wells 16and aspiration holes 22 in fluid communication, as shown in FIG. 2. Eachof the channels 28 is preferably roughly elliptical in shape due to thepreferred method of manufacturing used to create the channel, as will bedescribed hereinafter. Each of the channels is disposed beneath a bottomportion of one of the wells 16 and the aspiration holes 22, also due tothe preferred method of manufacture. Many types of shapes could be usedfor the channel 28 as the pressure distribution of the vacuum applied bythe pipette 34 will still be evenly distributed throughout the cell wellbeing aspirated. Each of the channels 28 acts, along with its respectiveone of the wells 16 and the holes 22, as a reservoir for excess media 36that has not been wicked into the frit 30. In addition to supplying avacuum for aspiration, the channels 28 and aspiration holes 22 couldalso be used to supply media 36. Supplying media through the channels 28and aspiration holes 22 to the wells 16 could be useful, for instance,if the callus of the plant tissue 32 was dense enough to be relativelyimpermeable to media dispensed into through the upper edge of the wells16. Preferably, the width of each of the channels 28 is less than thediameter of its respective one of the wells 16 so as to form a ledge 29at the bottom of the well for supporting the frit 30 above the media 36.

Each frit 30 is preferably constructed of a sintered polyethylenematerial that is porous and hydrophilic to promote the attraction andretention of the media within its interstices. The hydrophilicity of thefrit material can be permanent or temporary depending upon the processesby which it is applied, or whether the material is inherentlyhydrophilic. Alternative materials with porous structures could be usedand a surfactant could be applied to materials not naturally hydrophilicto make them hydrophilic. The material is produced by sintering andwicks solutions by capillary action and can act as a sterile barrier dueto its tortuous path properties. The frits are preferably cut or punchedin disk shapes roughly congruent with the wells 16 from porous ¼ inchthick polyethylene sheet with an average pore size of 90 to 130micrometers known as Porex from Porous Products of Fairburn, Ga. andalso available as Part No. Y2-PEH-250/90 from Small Parts Inc., MiamiLakes, Fla. Such a frit can hold about 550 μl of media and supply thetissue for a number of days.

The congruency of shape between the frit 30 and its respective one ofthe wells 16 ensures a fit with minimal leakage of the media 36 aroundthe frit and also ensures that the frit rests firmly on the ledge 29,above most of the media. Different thicknesses of the frit 30 may beused, with thicker frits generally holding more material and needing tobe refreshed with new media less often. A larger diameter frit could beused for larger diameter wells 16, but may require a manifold underneathfor additional support to keep the large diameter frit 30 fromcollapsing under the applied vacuum pressure. For instance, a largediameter frit 30 could be supported by a screen disposed on the back ofthe frit material when the frit material is manufactured. Despite thepossible need for a supporting manifold, the frits are typically muchstronger than similarly sized membranous or paper supports which cannotwithstand even moderate vacuum pressures applied by the pipette tipduring aspiration. Preferably, the frit material of the presentinvention should be able to withstand pressures of about 30 inches ofHg. In yet another embodiment, the tissue 32 could be grown on a sheetof the frit material, and then cut or punched into individual frits forplacement into the wells 16.

The MWP 10 is preferably constructed from a block of polycarbonatematerial of roughly the same rectangular dimensions as the body 12.Constructing the plate from a single block of material insures againstleakage between wells which could result in cross-contamination of thesamples. The wells 16 and aspiration holes 22 are preferably formedusing a CNC, or other automated drilling machine, with drill bits ofsimilar dimensions to the desired hole dimensions. If necessary, thedrilling machine also cuts away enough polycarbonate to form the base13, the inset above the base, the chamfers 15 and removes enoughmaterial to flatten out other surfaces, such as the upper surface 14.The channel 28 is preferably formed using a rotating saw blade 80 on thesame milling machine. The rotating saw blade has a cutting diameter ofslightly less than the wells 16 and is inserted into one of the wellsuntil it cuts away to a depth equal to the thickness of the rotating sawblade 80, as shown in FIG. 6. The rotating saw blade is then advanced inthe direction of the adjacent one of the aspiration holes 22 until itcuts into the aspiration hole enough to form the channel 28 withsufficient size to enable fluid communication between the well and theaspiration hole, as shown in FIG. 2. The saw blade 80 is then moved backto the center of the well and retracted out of the well. The undersizeddiameter of the saw blade forms the ledge 29 on which the frit 30 issupported.

As shown in FIGS. 2 and 3, the present invention can also include a seal35 that embraces the pipette tip 34 and is disposed around the upperedge 24 of one of the aspiration holes 22 when the tip is insertedtherein. The seal 35 includes an elastomeric ring 50 subjacent a rigidcollar 51. The rigid collar is preferably constructed from a stiffmaterial, such as from a steel bushing, and optionally includes a setscrew 52 extending through its side. Tightening of the set screwtightens the collar about the pipette tip 34 allowing the rigid collar51 to prevent upward migration of the elastomeric ring 50 as it ispressed against the upper edge 24 of the aspiration hole. The end of theelastomeric ring 50 that makes contact with the upper edge 24 of theaspiration hole can have a frustoconical shape with a preferred angle ofabout 70° to further facilitate formation of a vacuum-tight seal.Further preferably, the seal 35 is approximately 0.3 inches in diameterand 0.5 inches long so as to fit a standard pipette tip. For instance,TECAN pipette tip No. 71-700S with a PTFE (TEFLON) coating, an insidepoint diameter of 0.5 mm, outside point diameter of 1.1 mm, inside bodydiameter of 1.5 mm and an outside body diameter of 2.0 mm.

The elastomeric ring 50 may also be configured to fit any type ofpipette tip by sizing its aperture 53 to be about 90% of the widestoutside diameter of the pipette tip, allowing the seal to compressaround the tip while relaxing to its normal shape when removed from theaspiration hole. Conversely, the rigid collar 51 has an aperture that isoversized 10% with respect to the outside diameter of the pipette tip 34allowing it to easily receive the pipette tip. The collar's aperture isdecreased when the set screw 52 is tightened to secure the collar aboutthe pipette tip 34. In an alternative embodiment, the seal 35 could alsobe integrally molded with the pipette tip 34. In yet another embodiment,a soft sealing material could be used around the upper edge 24 of theaspiration holes so as to sealingly receive a tip without the seal 35.

The MWP 10 is used to promote tissue growth and propagation by supplyingnutrients in a sterile environment. The method for using the MWPincludes loading the plate either manually, or using a cell-sortermodified to sort tissue samples, such as those used to sort fruit flies.Generally, this equipment uses a vacuum to pick up the samples. Theunmodified cell sorter could be used if the tissue samples are smallenough. The MWP 10 is preferably covered with a transparent, polystyrenecover or lid to allow the transmission of light to the tissue.

Once the tissue 32 is in the wells 16 of the plate, the plate is stackedfor access by a liquid handler including multiple pipette tips connectedto media and vacuum supplies. The liquid handler grips the plate andremoves the lid in a manner known to those of skill in the art. Theliquid handling device extends each pipette tip 34 into a respective oneof the aspiration holes 22 until the seal 35 abuts the upper edge 24 ofthe aspiration hole and the portion of the upper surface 14 thereabout.The liquid handling device dispenses media through the tips, into theaspiration holes 22, through the channels 28 and into the wells 16. Inthis manner, the tissue 32 in each of the wells 16 has access to asupply of the media through the frit 30. The lid is replaced on theplate 10 and the plate is placed in a culture room with illumination topromote growth (in the case of plant tissues).

After the tissue depletes essential nutrients in the media 36, or themedia otherwise needs to be changed, the plate 10 is loaded back on theliquid handling device. The lid is removed from the plate. The liquidhandling device extends each pipette tip 34 into a respective one of theaspiration holes 22 until the seal 35 abuts the upper edge 24 of theaspiration hole and the portion of the upper surface 14 thereabout. Theliquid handling device applies a vacuum or suction pressure through thepipette tips which draws the media from the cell wells 16, through thechannels 28, into the aspiration holes 22 and into the pipette tips tocomplete aspiration. The media is cycled as often as needed by repeatingthe above process.

In another embodiment, an automated pipetting head (or robotic liquidhandler) 60 is used to address single or multiple wells selectively,such as the GENESIS liquid handler (TECAN, AG of Switzerland), shown inFIG. 4. The robot is upgraded with a six-way valve for switching betweendifferent media, reagent and ethanol supplies. The robot includes eightpipetting tips 61 on its liquid handling arm 62. Four of the tips areconfigured to deliver media to a plurality of multiple well plates 63supported on its deck 64. The four remaining tips are each fitted with atip seal allowing the four remaining tips to aspirate the wells 16through their respective aspiration holes 22.

Preferably, the robotic liquid handler 60 is operated using a softwareprogram to control deployment of its pipetting tips 61 in such a way asto minimize cross-contamination between the wells 16. During mediadelivery, contamination of the four media supplying tips is avoided bysuspending the media supplying tips over the wells without contactingthe plates. Contamination of the aspiration tips is avoided by flushingeach tip with an anti-microbial ethanol liquid in between aspirationcycles. The exterior of the tips and seals are washed in an on-deckshallow wash station 65, also filled with ethanol liquid. The ethanol ispumped by a syringe pump and/or a high-speed diaphragm (fast wash) pumpfrom a reservoir accessible through the six way valve.

Programming the robot 60 to access the correct holes requires teachingthe robot the location of both the aspiration holes 22 and the wells 16.In this manner, the robot is operated as if the plate 10 has twice asmany wells because the aspiration holes 22 are positioned to correspondto the wells of a plate having twice the density. The fact that theaspiration holes have a smaller diameter than the wells 16 is of noconsequence as the robot seeks the center of the aspiration holes 22.For instance, the robot is programmed to access a 24 well plate as if ithad 48 wells, each well having a diameter equal to that of theaspiration holes. Notably, the programming of the well centers (or theirlocations with respect to the aspiration holes) can be considerably offbecause of the much smaller diameter of the pipette tips 61 with respectto the wells.

FIG. 5 depicts one example of how the robot 60 can be programmed toaddress the wells 16 and aspiration holes 22 of the plate 10. First, thefour media supply tips are positioned above wells 105 through 108 and,without contacting the plate 10, dispense media into the wells in step125. The media supply tips are then sequentially positioned above, anddispense media into, wells 113 through 116 and 121 through 124 in steps126 and 127, respectively. The four aspiration tips are then insertedinto the aspiration holes (which the robot recognizes as “wells”) 101through 104, aspirating spent media from wells 105 through 108, in step128. After aspiration, the aspiration tips are flushed internally andcleansed externally with ethanol at the wash station 65, in step 129.Aspiration and cleaning are alternated for holes 109 through 112, insteps 130 and 131, and again for aspiration holes 117 through 120 insteps 132 and 133. It should be noted that the process can be expandedor reduced depending upon the number of pipetting tips and the number ofwells. In addition, the process is preferably performed within a HEPAfiltered environment, such as within a pressurized and filtered hood, tominimize the occurrence of contamination.

The robot 60 can also be used in conjunction with a high-density hotelor rack for holding and supplying light to the tissue in hundreds of theplates, as described in commonly owned U.S. patent application Ser. No.10/080,918 entitled, “LED Array for Illuminating Cell Well Plates andAutomated Rack System for Handling the Same,” which is incorporatedherein by reference. The automated rack system uses its own robot tomanipulate and present the plates to the robot of the present system.

In yet another embodiment, the well plate and method of the presentinvention could be used to perform solid phase extraction. Each well ofthe well plate contains a matrix trapped between a pair of frits whichform a column. Various compounds (such as a ligands, or antibodies) areforced through the column and become trapped within the matrix. Aftersolid phase extraction, an agent can be forced through the column tobreakup the solid phase. High-throughput distribution and retrieval ofthe compound, agents, etc. could be handled by an automated pipettinghead using the wells and their respective aspiration holes.

The present invention has several advantages. For example, the tissuesamples 32 in the wells 16 do not have to be moved or disturbed, cuttingdown on workload, ensuring sterile and optimal growth conditions. Theplates 10 may be used with conventional liquid handling pipette headsbecause the aspiration holes 22 are accessible from the upper surface ofthe body, i.e., a top-loading arrangement. The use of robotic liquidhandlers with the plate promotes a well-to-well consistency in thetreatment of the tissue, as well as the efficient removal andreplacement of the media 36. The top-loading aspect allows the use of astandard lid for sterility control and removes the need for a separatevacuum manifold station for pulling out media. The lack of a manifoldallows for the differential treatment of each well and providesflexibility in liquid handler design and selection. The liquid head canbe configured to remove the media as well as add new media with nochange of tooling or pipette tips.

Some of the figures disclosed herein contain block diagrams, flowchartsand control flow illustrations of methods, systems and program productsaccording to the invention. It will be understood that each block orstep of the block diagram, flowchart and control flow illustration, andcombinations of blocks in the block diagram, flowchart and control flowillustration, can be implemented by computer program instructions. Thesecomputer program instructions may be loaded onto a computer or otherprogrammable apparatus to produce a machine, such that the instructionswhich execute on the computer or other programmable apparatus createmeans for implementing the functions specified in the block diagram,flowchart or control flow block(s) or step(s). These computer programinstructions may also be stored in a computer-readable memory that candirect a computer or other programmable apparatus to function in aparticular manner, such that the instructions stored in thecomputer-readable memory produce an article of manufacture includinginstruction means which implement the function specified in the blockdiagram, flowchart or control flow block(s) or step(s). The computerprogram instructions may also be loaded onto a computer or otherprogrammable apparatus to cause a series of operational steps to beperformed on the computer or other programmable apparatus to produce acomputer implemented process such that the instructions which execute onthe computer or other programmable apparatus provide steps forimplementing the functions specified in the block diagram, flowchart orcontrol flow block(s) or step(s).

Accordingly, blocks or steps of the block diagram, flowchart or controlflow illustration support combinations of means for performing thespecified functions, combinations of steps for performing the specifiedfunctions and program instruction means for performing the specifiedfunctions. It will also be understood that each block or step of theblock diagram, flowchart or control flow illustration, and combinationsof blocks or steps in the block diagram, flowchart or control flowillustration, can be implemented by special purpose hardware-basedcomputer systems which perform the specified functions or steps, orcombinations of special purpose hardware and computer instructions.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing descriptions andthe associated drawings. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims. For instance, in anotherembodiment the well plate and method could also be used to growbacterium. Although specific terms are employed herein, they are used ina generic and descriptive sense only and not for purposes of limitation.

1. A method of making a plate for holding a porous frit that supports atissue, the porous frit and the tissue bathed in a media that isregularly aspirated and refreshed by a pipette device, said methodcomprising: providing a body with an upper surface; defining a firsthole in the body by drilling through the upper surface of the body toform a first hole upper edge and drilling below the upper surface andinto the body to form a first hole bottom portion; defining a secondhole in the body by drilling through the upper surface of the body toform a second hole upper edge and drilling below the upper surface andinto the body to form a second hole bottom portion; and connecting thefirst hole bottom portion to the second hole bottom portion by forming apassage in the body and between the first hole bottom portion and thesecond hole bottom portion.
 2. A method of making the plate of claim 1,wherein forming the passage includes inserting a saw into the first holebottom portion and moving the saw laterally until encountering thesecond hole bottom portion.
 3. A method of making the plate of claim 1,wherein the body is defined from a single piece of material.